Abstract

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291–298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1 mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1 mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H₂O₂) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox^® (a water soluble Vitamin E analogue) conferred significant protection (P < 0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days’ supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P < 0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Keywords: Buccal cells; Comet assay; DNA damage; Oxidative stress; Antioxidant; Genoprotection

Article Outline

1. Introduction

2. Materials and methods

2.1. Reagents

2.2. Buccal cell collection technique

2.3. Method development

2.3.1. Cell lysis

2.3.1.1. Effect of detergents

2.3.1.2. Effect of trypsin and proteinase K

2.3.2. Electrophoresis

2.4. Method evaluation

2.4.1. Effect of oxidant challenge (induced by H₂O₂)

2.4.2. Effect of in vitro antioxidant exposure

2.4.3. Biological variation and effect of in situ antioxidant (green tea) exposure

2.4.4. Effect of ‘in vivo’ antioxidant (Fructus barbarum L.) supplementation

2.5. Statistical analysis

3. Results

4. Discussion

Acknowledgments

References